Collaborative vaccine clinical trials are now ongoing at the NIH and numerous Cancer Centers throughout the U.S. with vaccines developed in this program. The tumor-associated antigens targeted by these vaccines are (a) carcinoembryonic antigen (CEA), which is overexpressed on the majority of human carcinomas, (b) MUC-1, which is overexpressed on breast and other carcinomas, and (c) prostate-specific antigen (PSA). The genes for these tumor-associated antigens have been placed into two types of recombinant orthopox vectors: recombinant vaccinia and the replication defective avipox virus. Preclinical studies and clinical trials have now demonstrated the optimal use of these vectors in diversified prime and boost vaccine protocols. Immunoassays have been and are currently being developed which detect and characterize T-cell responses to these tumor-associated antigens in vaccinated patients. It has now been shown that vaccinated patients can mount specific immune responses to known epitopes on these tumor antigens; the T cells generated can in turn kill tumor cells expressing these antigens. Agonist epitopes have now been identified for both CEA and PSA. Single amino acid changes in these epitopes have been shown to enhance the generation of cytolytic T-cell responses to both CEA and PSA expressing tumor cells. Previous studies have shown that a specific 9-mer amino acid epitope (designated CAP-1) of the human "self" tumor-associated carcinoembryonic antigen can be used to stimulate CD8+ T cells from peripheral blood mononuclear cells of carcinoma patients vaccinated with pox vector-based carcinoembryonic antigen vaccines. A T-cell receptor agonist epitope of CAP-1 (designated CAP1-6D) has been shown to enhance the stimulation of T cells over levels obtained using CAP-1. We analyzed gene expression profiles in T cells stimulated with the native CAP-1 versus the agonist CAP1-6D peptide. Microarray analyses were conducted to analyze differential gene expression profiles of a T-cell line stimulated with native versus agonist peptides. Numerous genes and gene clusters are identified as differentially expressed as a consequence of stimulation with the agonist peptide versus the native peptide; two genes, however, stand out in magnitude: the chemokine lymphotactin and granzyme B. In particular, lymphotactin expression is >12 times more pronounced in agonist-stimulated T cells. An ELISA assay was developed that confirmed marked lymphotactin secretion in T cells when stimulated with the agonist versus the native peptide. A chemotaxis assay also demonstrated the biological activity of the lymphotactin produced. To our knowledge, these are the first studies of gene expression profiles of a defined T-cell line in response to stimulation with a defined antigen. They are also the first to compare, via cDNA microarray, responses of a T-cell line to (a) a tumor-associated self-antigen and (b) a native epitope versus an agonist epitope.Dendritic cells (DCs) are the most potent of the antigen-presenting cells (APCs). Preparation of sufficient numbers of mature DCs, however, is both costly and time-consuming. We have examined here the possibility of using an alternative source of APCs that would be easier to obtain, would not require extensive culture, and thus would be more applicable to human immunotherapy protocols. We have shown that freshly isolated human B cells can be efficiently infected by a replication-defective fowlpox recombinant vector, designated rF-TRICOM (TRIad of COstimulatory Molecules), to markedly increase surface expression of the human costimulatory molecule B7-1 and moderately increase expression of intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-3 (LFA-3). Peptide-pulsed rF-TRICOM-infected B cells were highly efficient in activating antigen-specific human T cells and shown to be superior to the use of CD40L in enhancing APC potency. Moreover, when infection of freshly isolated B cells with rF-TRICOM was combined with CD40L, a still further marked enhancement of the antigen-presenting potency was observed. Ex vivo-generated antigen-specific T cells activated in this manner might be applied to experimental protocols or used for adoptive transfer in immunotherapy protocols.